Amylose acetate

ABSTRACT

A novel process for providing surgical hemostasis. In the process, an acetylated amylose with a critical range of acetate is employed.

United States Patent [191 Usher 1 1 Oct. 16, 19 73 [54] AMYLOSE ACETATEFOREIGN PATENTS OR APPLICATIONS 1 lnvemori Francis Usher, Houston900,868 7/1962 Great Britain 128/325 [73] Assignee: International PaperCompany,

New York, N.Y. Primary Examiner-Charming L. Pace I Attorney-Louis F.Reed [22] Filed: May 14, 1971 [21] Appl. No.: 143,616

[57] ABSTRACT [52] US. Cl 128/325, 424/15, 424/180,

123/ R A novel process for providing surgical hemostasis. In [51] 1111-(1- 1!) /1 A 1) /0 1 /0 the process, an acetylated amylose with acritical [58] Field of Search 128/1 R, 156, 296, range of acetate isemployed.

[56] References Cited 5 Claims, 0 ings AMYLQSE ACETATE DESCRIPTION OFTHE PRIOR ART Amylose acetate is an ester of amylose, the linearfraction of starch. Amylose comprises about 25 percent of the wholestarch. The acetate is obtained by acetylation of amylose with aceticanhydride as described, for example, by Jeanes et al. in the Journalofthe American Chemical Society, Vol. 74, pages 6116-6117 (1952) or byWolff et al. in the Journal of the American Chemical Society, Vol. 13,pages 346-349 1951). A fully acetylated amylose, viz. amylosetriacetate, has a D.S. (degree of substitution) of 3 which is equivalentto a theoretical acyl content of 44.8%. D.S. can be readily deter- Inthe above equation, 43" represents the molecular weight of the acetylgroup; 162" represents the molecular weight of the anhydroglucose unit.

Films of amylose triacetate are disclosed by Whistler et al. in theJournal of the American Chemical Society, 65, page I436 I943). Wolff etal. in Industrial and Engineering Chemistry, 49, pages 1247-1248 (Aug.1957) discloses films of both amylose triacetate and mixed esters ofamylose.

Amylose acetates of varying degree of acetate substitution are known.Thus, Cowie et al. in Die Makromolekulare Chemie, Vol. 121, pages 51-57(1969) disclose amylose acetates having a D.S. of 1.6 and 3.0. Muetgurtet al. in Die Starke, Vol. l0, pages 303-304 (1958) disclose a partiallyacetylated amylose. Wagner et al. in the Journal of the AmericanPharmaceutical Association, Vol. XLVlll, No.4, pp. 244-249 (Apr. 1959)disclose amylose acetate phthalates useful in enteric coatings. Inpreparing the amylose acetate phthalates, amylose is acetylated to aD.S. of 1.1 l to 2.67 and then reacted with phthalic anhydride.

Materials used today for applying to open wounds and incisions areprepared from heterologous protein as the submucosal layer of theintestine of sheep (catgut) or the Achilles tendon obtained from cattle(soluble collagen). Oxidized cellulose is also employed. These materialsact as foreign bodies and can cause considerable inflammatory reactionin tissues. Mihaly Gerendas, in a book entitled Fibrinogen, edited byKolomani Laki, published in 1968, discloses that inadequate hemostasisrepresents a problem, not only in cases of clotting disorders, but oftenin normal surgical procedures because diffuse, parenchymatous bleedingis very troublesome. Fibrinogen and thrombin preparations in differentforms are suggested to aid hemostasis.

British patent 900,868 discloses amylose sponges and gauze. U.S. Pat.No. 3,499,074 produces amylosic starch filaments from an aqueousdispersion of amylosic solids," which is defined as any mixture offilament-forming solids which includes at least about 50 percent byweight of pure amylose or an amylose derivative such as amylose ether,ester or anhydride. The

and can be digested by humans and other living systems, and thus satisfythe requirements for use in specialized applications as medical suturesor bandages. A hydroxypropyl derivative of a high amylose starch mixedwith 4 percent glycerine and 26 percent water is specifically disclosedin the patent for forming amylosic monofilaments. Other patentsconcerned with wound treating are U.S. Pat. No. 3,328,259 which showsthe use of a film containing a water-soluble cellulose compound topromote coagulation and U.S. Pat. No. 3,470,295 which discloses the useof maltose, inositol, and sorbitol in the treatment of humans andanimals for hemorrhage.

The prior art relating to wound application suffers from a number ofdisadvantages. These include inefficient hemostasis, for example,because of absorption of the applied agent too quickly or poorabsorption of the applied agent, intorduction of foreign bodies, toomuch tissue response, and/or high expense.

SUMMARY OF THE INVENTION It is an object of the invention to provide anefficient process for surgical hemostasis.

Another object of the invention is to provide an economical process forproviding surgical hemostasis using a material that is digested byenzymes, as amylase and diastase, present in the tissue fluids of humansand animals.

Still another object of the invention is to provide a process whichstops hemorrhaging of a wound within a few minutes after application.

It is still another object of the invention to provide a hemostaticsurgical product which is not absorbed by the body too quickly to loseits effectiveness but is absorbed in the body in a short period ofwithin a matter of only a short number of days, usually 3 or 4 days,with minimal tissue response and without tending to cause infectionswhich prior art hemostatic agents tend to cause. This permits the use oflarge amounts of the hemostatic surgical product of the invention sinceit is sufficiently rapidly absorbed by the body without untowardresults.

Another object of the invention is to provide an absorbable hemostaticsurgical product which degrades into glucose, a normal constituent ofhuman and animal tissue fluids, and small amounts of acetic acid thatcause little or no tissue reaction.

It is yet another object of the invention to provide an absorbablehemostatic surgical product which is supple and elastic duringapplication to ensure good contact with a profusely bleeding wound andpromote hemostasis.

A further object of the invention is to provide an absorbable hemostaticsurgical product that sticks tenaciously to the wound and/or surroundingtissue.

It is also an object of the invention to provide an absorbablehemostatic surgical product that is easily and reliably sterilized.

Briefly the present invention concerns depositing on the surface of awound, incision or lesion a partially acetylated amylose with a criticalacetate range.

DETAILED DESCRIPTION OF THE INVENTION According to the presentinvention, partially acetylated amylose having a D.S. of between about0.2 and about 1.5, preferably between about 0.2 and 0.6, is applied tothe surface of a wound, incision or lesion. In application, the amyloseacetate is desirably inthe form of a film, preferably one of about 1 to4 mils thick and desirably having a number of perforations in the film.Thicker films are less desirable since they have less tendency toconform to the surface of the wound and may not provide intimatecontact. The surface being treated need not be completely covered withthe amylose acetate, although it is desirable that the extent of thewound, incision or lesion be covered by the amylose acetate and that thefilm cover at least some of the adjacent surrounding tissues. Ifdesired, more than one sheet of a perforated film may be superimposedover the wound, etc., or parts of two or more films of amylose acetatemay overlap.

For obtaining the amylose acetate, amylose is desirably acetylated in aheated formamide solvent, using sodium acetate as the catalyst, inreaction with acetic anhydride. The procedure is derived from A. Jeanesand R. Jones as published in Journal ofAmerican Chemical Society, Vol.74, page 61 16 (1952). By controlling the amount of acetic anhydrideused, amylose acetates containing the critical acetate range (degree ofsubstitution), are obtained.

Amylose acetate films can be made by casting directly the amyloseacetate from a filtered, deaerated formamide solution of the amyloseacetate. Concentrations of about 20 percent by weight of amylose acetateare desirable. Alternatively, amylose acetate from a filtered, deaeratedformamide solution is precipitated in water or acetone, then redissolvedin dimethylsulfoxide, the solution deaerated and films cast therefrom.

The films are cast by drawing the amylose acetate solution onto highlypolished ferrotype plates which had been sprayed with silicone releaseagent. Cast films are regenerated by placing the plates in water. Thefilms may be dried in the oven at 65C. and stripped from the plates.

For application to a wound, incision or lesion a. respective film isperforated, sterilized by chemical sterilizing agents, such as gassterilization, with ethylene oxide at a temperature of about 80-100F.for several hours, and then placed on a wound. The films arc desirablyperforated with small holes so as to prevent the entrapment of airbubbles below the surface of the film of amylose acetate. The films areheld in place for a few minutes during which time they form a sticky,elastic coating which sticks tenaciously to the wound and stops anybleeding. If bleeding is not stopped with sufficient rapidity, anadditional perforated film may be placed over the first film. Iodinestaining readily indicates that the film has been absorbed.

Further details of the invention are illustrated by the followingexamples which represent various embodiments. All proportions given areby weight unless otherwise indicated.

EXAMPLE 1 a. Preparation of amylose acetate films i. Ninety grams ofSuperlose, a 95% amylose starch fraction, were mixed in 400 millilitersof formamide at 23C. for 30 minutes in a one-liter resin kettle. Themixture was heated to 95-100C. by placing the resin kettle in a hotwater bath. it took 30 minutes to reach 98C. grams of anhydrous sodiumacetate were added to the mixture and the mixture mixed at 98C. for 30minutes. ()ne hundred milliliters of acetic anhydride were then addeddropwise to the mixture over'a 15 minute period. The reaction wasstirred an additional minutes at 98C. with the reaction proceeding asevidenced by the amylose going into solution. Over the next 60 minutes,the mixture cooled to 30C. 100 milliliters of formamide and 10milliliters of water were subsequently added and the mixture stirred for30 minutes. The solution was filtered through a dacron cloth on aBuchner funnel to remove grit and the solution deaerated in a vacuumdesiccator. I Films of the amylose acetate dissolved in an amount ofabout 20 percent by weight in formamide were cast on KindermannFerrotype plates treated with a silicon spray release agent and using aBoston Bradley draw down bar with clearance adjusted with a microscopeglass slide. The plates were then placed in the oven at C. The plateswith the adhering films were conditioned at room temperature for atleast 15 minutes and then the films were stripped by hand from theplates and trimmed. The films were prepared having a thickness ofbetween about 1 and 4 mils. The films were regenerated by placing thedrawn liquid films in distilled water overnight.

In this manner, a film (186 JM 45-A) of amylose acetate with a D5. of0.906 was prepared.

ii. In an alternative procedure, 200 milliliters of the i deaerated,filtered formamide solution from (i) were poured slowly into 1200milliliters of acetone and an additional 800 milliliters of acetone wereadded. The amylose acetate precipitate settled and the clear liquiddecanted off. The precipitate was mixed twice, each time with 1000milliliters of acetone, and the clear liquid decanted off. Theprecipitate was then recovered by filtration, washed with 300milliliters of acetone and pressed. Yield was 73.0 grams wet (3.6 gramswet is equivalent to 1.60 grams dry).

Dried powder was dissolved in dimethylsulfoxide (DMSO) to form a 15percent solution. The solution was cast as in (i). In this manner, afilm of amylose acetate with 0.654 D.S. was prepared.

iii. In yet a third procedure, 200 milliliters of the deaerated,filtered formamide solution from (i) were poured slowly into 2000milliliters of water. The amylose acetate precipitate settled and theclear supernatant liquid decanted off. The precipitate was washed fourtimes, each time with two liters of water, then filtered and pressed.The powder, slurried with a minimum amount of water, was put into freezedry bottles and freeze dried to yield a fine powder. The powder can thenbe used to provide films cast as in (ii) from a 20 percent solution ofthe powder in DMSO.

Films produced in accordance with this and the other examples hereinwere gas sterilized prior to use by first evacuating a chambercontaining the perforated films and then introducing ethylene oxide gasinto the chamber and maintaining the chamber and its contents at atemperature of F. for 3 hours at a slightly elevated pressure.

b. Hemostatic Action of Films A tangential section of the liver ofa dog(animal No. 8552) was excised (about 1 X 2 inches) and two (2) sheets ofamylose acetate film of about 2-3 mils thickness with a D.S. of 1.25,produced in a manner substantially similar to part (i) of this example,were applied over the wound. Bleeding immediately stopped. The

. same procedure was done on the dogs spleen. Bleeding immediatelystopped, although a somewhat longer time was taken for the hemostasis.

An autopsy of the dog 3 days later showed no blood in its abdomen. Also,there were few omental adhesions to the spleen and none to the liver.Cut surfaces of the liver and spleen were free of blood clots. There wasno evidence of the applied film, it having been absorbed by the dogstissue fluids. Both cut surfaces were smooth and appeared wellorganized.

EXAMPLE 2 A midline incision on a dog (animal No. 8269) was made. Onelarge piece was cut from the spleen. Amylose acetate film of D.S. 0.906(produced in accordance with Example la(i)) was applied to the bleedingsurface. The bleeding stopped in 10 to 15 minutes. The dog was thenclosed and given penicillin and streptomycin. An autopsy of the dog 6days later showed no blood in the abdomen.

A section of the rectus muscle of the same dog was excised and anotheramylose acetate film of D.S. 0.906 applied to the cut muscle surface.The bleeding stopped.

A section of the spleen of the same dog was excised and amylose acetatefilm of D.S. of 0.906 was applied. The bleeding stopped.

EXAMPLE 3 A midline incision on a dog (animal No. 8627) was made. Two(2) pieces were excised from the liver and spleen and replaced withperforated amylose acetate film (about 23 mils thick) of D.S. 0.906. Thebleeding stopped in l to l5 minutes. The wound was closed and the doggiven penicillin and streptomycin. An autopsy of the dog 6 days latershowed no blood in the abdomen.

The liver of the same dog was cut and another film of amylose acetate ofD.S. 0.906 was applied over the wound. The bleeding stopped. Iodine testfor starch made 6 days later on a healthy surface of the spleen wasnegative for starch.

EXAMPLE 4 A midline incision on a dog (animal No. 660) was made. Therecti sheaths on each side of the midline were opened and the rectimuscles split. Each incision was 8 inches long. On the left side a pieceof amylose acetate film of D.S. 0.26 (produced in accordance withExample la(i), but using less acetic anhydride) measuring 6 X 1 inch(perforated film of 23 mils thick folded four times) was implantedbetween the recti muscles and transversalis fascia. The rectus fascia(interior) was closed with three-strand coated wire suture. On the rightside (control), no implantation was made, although the same type ofclosure was made. Pencillin and streptomycin were given to the dog.

An autopsy of the dog three days later showed no visible trace of theamylose acetate film previously implanted deep into the rectose muscle.The wound was repeatedly swabbed with saline moistened gauze sponge totest for free starch 6 separate sponges being used. No trace of starchwas found in the wound. The Combistix test strip showed no free glucosepresent. Thus the amylose acetate had been completely absorbed. Thecontrol wound had the same appearance as the implanted wound.

EXAMPLE 5 A midline incision, 10 inches long, was made on a dog (animalNo. 720). Then a rectus sheath on both sides was incised for 8 inchesand both recti muscles split longitudinally for 8 inches. A 6 inch rollof amylose acetate film of D.S. 0.32 (about 4 to 6 folds) was implantedin each incision. The rectus sheath was then closed with running7-strand No. 0 coated wire suture. Then the subcutaneous space wasfilled with amylose acetate film of D.S. 0.32 for 10 inches length and 2inches width. About 6-8 folds of amylose acetate film were used. Thenthe subcutaneous tissue was closed over the film using 7-strand No. 0suture. The skin was closed with running silk. Penicillin andstreptomycin were given to the dog for three days.

An autopsy of the dog seven days later showed upon repeated tests withtincture of iodine that the subcutaneous space and both intra-muscularspaces were negative for starch. The Combistix test for glucose was alsonegative. There was no visible evidence of any amylose acetate film.Thus the amylose acetate had been completely absorbed'by the dogstissues.

EXAMPLE 6 A midline incision 10 inches long was made on a dog (animalNo. 936). A rectus sheath on both sides was incised for 8 inches andboth recti muscles split longitudinally for 8 inches. A 6 X 1 inch film(flat) of amylose acetate of D.S. 0.32 was implanted deep to the rectimuscles on each side. The rectus sheath was then closed with suture.Then one sheet 6 X 2 inches of amylose acetate of D.S. 0.32 was placedsubcutaneously and the subcutaneous tissue closed with the suture. Theskin was also closed with the same suture and the dog given penicillinand streptomycin.

An autopsy of the dog one day later showed serum (pink-tinged) presentin the subcutaneous space and in both intra-muscular spaces. There wasno infection. Repeated tests with tincture of iodine in the subcutaneousspace and both intra-muscular spaces were negative for starch. There wasno visible evidence of the amylose acetate film, it having been absorbedby the animals tissues. Combistix tests for glucose were also neg ative.No hematoma was present.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed.

What is claimed is:

l. A process for providing surgical hemostasis at the site of a woundwhich comprises applying to the surface of the wound amylose acetatehaving a degree of substitution of between about 0.2 and 1.5.

2. A process in accordance with claim 1 wherein amylose acetate having adegree of substitution of between about O.2 and 0.6 is applied to thesurface of the wound.

3. A process in accordance with claim 1 wherein there is applied to thesurface of the wound a film of amylose acetate having a degree ofsubstitution of between about 0.2 and 1.5.

4. A process in accordance with claim 3 wherein there is applied to thesurface of the wound a film of between about 1 and 4 mils thick ofamylose acetatehaving a degree of substitution of between about 0.2 and1.5.

5. A process in accordance with claim 3 wherein there is applied to thesurface of the wound a perforated film of amylose acetate having adegree of substitution of between about 0.2 and 1.5.

2. A process in accordance with claim 1 wherein amylose acetate having adegree of substitution of between about 0.2 and 0.6 is applied to thesurface of the wound.
 3. A process in accordance with claim 1 whereinthere is applied to the surface of the wound a film of amylose acetatehaving a degree of substitution of between about 0.2 and 1.5.
 4. Aprocess in accordance with claim 3 wherein there is applied to thesurface of the wound a film of between about 1 and 4 mils thick ofamylose acetate having a degree of substitution of between about 0.2 and1.5.
 5. A process in accordance with claim 3 wherein there is applied tothe surface of the wound a perforated film of amylose acetate having adegree of substitution of between about 0.2 and 1.5.